Gene promoter sequences and uses thereof

ABSTRACT

The promoters of the  Capsicum annuum  (bell pepper) fibrillin gene has the nucleotide sequence SEQ ID NO: 1. This promoter is useful for driving expression of foreign genes in transgenic plants.

The present invention relates to gene promoter sequences isolated frombell pepper and their use to regulate chimeric gene expression inplants. In particular it describes the isolation and use of DNAsequences which permit a high level of expression of foreign genes intransgenic plants.

The expression of genes in plants is controlled by a number ofregulatory components, including nucleic acid and protein elements.Where the plant gene exists as double stranded DNA, the primary steps ofexpression involve the production of a messenger RNA by a polymeraseenzyme. The initiation of this part of the expression process iscontrolled by a region commonly referred to as the “promoter”. Thepromoter lies upstream (5′) of the protein encoding region and may beconstitutive or tissue-specific, developmentally-regulated and/orinducible.

Within the promoter region there are several domains which are necessaryfor full function of the promoter. The first of these domains liesimmediately upstream of the structural gene and forms the “core promoterregion” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence which defines thetranscription start point for the structural gene. The precise length ofthe core promoter region is indefinite but it is usuallywell-recognisable. Such a region is normally present, with somevariation, in all promoters. The base sequences lying between thevarious well-characterised “boxes” appear to be of lesser importance.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore. the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences, usually upstream ofthe core, constitute the remainder of the promoter. The regulatorysequences determine expression level, the spatial and temporal patternof expression and, for an important subset of promoters, expressionunder inductive conditions (regulation by external factors such aslight, temperature, chemicals, hormones). Manipulation of crop plants toalter and/or improve phenotypic characteristics (such as productivity orquality) requires the expression of heterologous genes in plant tissues.Such genetic manipulation therefore relies on the availability of meansto drive and to control gene expression as required; for example, on theavailability and use of suitable promoters which are effective in plantsand which regulate gene expression so as to give the desired effect(s)in the transgenic plant. It is advantageous to have the choice of avariety of different promoters so that the most suitable promoter may beselected for a particular gene, construct, lo cell, tissue, plant orenvironment.

Promoters (and other regulatory components) from bacteria, viruses,fungi and plants have been used to control gene expression in plantcells. Numerous plant transformation experiments using DNA constructscomprising various promoter sequences fused to various foreign genes(for example, bacterial marker genes) have led to the identification ofuseful promoter sequences. It has been demonstrated that sequences up to500-1000 bases in most instances are sufficient to allow for theregulated expression of foreign genes. However, it has also been shownthat sequences much longer than 1 kb may have useful features whichpermit high levels of gene expression in transgenic plants. A range ofnaturally-occurring promoters are known to be operative in plants andhave been used to drive the expression of heterologous (both foreign andendogenous) genes in plants: for example, the constitutive 35Scauliflower mosaic virus promoter, the ripening-enhanced tomatopolygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology,11:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO,7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989,Plant Molecular Biology, 13:639-651) and many others.

As stated above, successful genetic manipulation relies on theavailability of means to control plant gene expression as required. Thescientist uses a suitable expression cassette (incorporating one or morepromoters and other components) to regulate gene expression in thedesired manner (for example, by enhancing or reducing expression incertain tissues or at certain developmental stages). The ability tochoose a suitable promoter from a range of promoters having differingactivity profiles is thus important.

In the present invention, we have isolated and fully sequenced thefibrillin gene promoter and the capsanthin-capsorubin synthase genepromoter from bell pepper (Capsicum annuum). The fibrillin promoter(FIB) essentially controls the production of the protein known as“fibrillin” in peppers. This fibrillin protein is associated with theplant chromoplasts and is involved in the packaging and organisation ofcarotenoids. It is the FIB promoter that regulates the accumulation offibrillin during chromoplast differentiation.

The capsanthin-capsorubin synthase promoter (CCS) controls theproduction of the enzyme capsanthin-capsorubin synthase. This enzymecatalyses the conversion of the ubiquitous 5,6-epoxycarotenoids,antheraxanthin and violaxanthin, into capsanthin and capsorubin,respectively. It is the CCS promoter that specifically regulates the CCSgene during chloroplast to chromoplast differentiation.

The present invention aims to provide, inter alia, alternative promoterscapable of driving gene expression in plants. We believe the presentinvention provides new developmentally regulated promoters which may beparticularly useful in controlling chimeric gene expression inparticular parts of a plant at a specific stage during development e.g.fruit ripening. This may be especially useful in plants such as tomatoplants.

According to the present invention, there is provided a DNA sequenceencoding a bell pepper fibrillin gene promoter capable of driving geneexpression in plants having the sequence shown in SEQ ID NO 1 or activevariants thereof.

Further according to the present invention, there is provided a DNAsequence encoding a bell pepper capsanthin-capsorubin gene promotercapable of driving gene expression in plants having the sequence shownin SEQ ID NO 2 or active variants thereof.

The cDNA sequence of the bell pepper FIB gene from the ATG initiationcodon to a position 214 bp upstream, and of the CCS gene from the ATGinitiation codon to a position 200 bp upstream have been previouslydescribed by Deruere et al (1994) (Biochem. Biophys. Res.Commnun. 199(3) 1144-50). Similarly, the cDNA sequence of the CCS gene from the ATGinitiation codon to a position 66 bp upstream has been described byBouvier et al (The Plant Journal 6 (1) 45-54). The invention does notextend to these DNA sequences per se but does cover their use in theconstructs, expression cassettes and the methods of the invention asdescribed further herein.

“Active variants” are DNA sequences partially homologous to SEQ ID NO 1or SEQ ID NO 2 which retain promoter activity. It may be possible toalter the level or type of activity of these promoters by manipulatingtheir sequences: for example, by altering the nucleotide sequence in keyregulatory regions, by truncating the sequence or by deleting partswithin the sequence.

The promoters of the invention are suitable for incorporation into DNAconstructs encoding any target gene or transcribable DNA region so thatthe target gene is expressed when the construct is transformed into aplant. The DNA construct preferably contains a transcription terminationsignal.

The bell pepper FIB and CCS promoters may be synthesised ab initio usingthe sequence shown in SEQ ID NO 1 and SEQ ID NO 2 as a guide.Alternatively, the promoters may be isolated from plant genomic DNAlibraries using suitable probes derived from the said sequences or thepromoter may be isolated using a PCR approach.

In practice the promoter of the invention may be inserted as a promotersequence in a recombinant gene construct designed for use in a plant.The construct is then inserted into the plant by transformation. Anyplant species may be transformed with the construct, and any suitabletransformation method may be employed. It is preferred that plants to betransformed with the promoters according to the present invention areplants containing chromoplasts.

According to a second aspect of the invention, there is provided a plantgene expression assette comprising the bell pepper FIB or CCS promoteroperatively linked to a target gene, the promoter having the sequenceshown as SEQ ID No 1, SEQ ID No 2 or active variants hereof.

The target gene is a DNA sequence which may be derived from anendogenous plant gene or from a foreign gene of plant, fungal, algal,bacterial, viral or animal origin. Normally it is a sequence other thanthe sequence encoding the FIB or CCS protein which follows the FIB orCCS promoter in the naturally occuring bell pepper FIB or CCS gene. Thetarget gene may be a single gene or a series of genes. The target geneis adapted to be transcribed into functional RNA under the action ofplant cell enzymes such as RNA polymerase. Functional RNA is RNA whichaffects the biochemistry of the cell: for example, it may be mRNA whichis translated into protein by ribosomes or it may be RNA which inhibitsthe translation of mRNA related to it. Thus the target gene sequence maybe a sense sequence encoding at least part of a functional protein or anantisense sequence.

The expression cassette is suitable for general use in plants. Inpractice the DNA construct comprising the expression cassette of theinvention is inserted into a plant by transformation. Any transformationmethod suitable for the target plant or plant cells may be employed,including infection by Agrobacterium tumefaciens containing recombinantTi plasmids, electroporation, microinjection of cells and protoplasts,microprojectile transformation, pollen tube transformation andtransformation of plant cells using mineral fibres (U.S. Pat. No.5,302,523, International Patent Application Publication NumberW094/28148). The transformed cells may then in suitable cases beregenerated into whole plants in which the new nuclear material isstably incorporated into the genome. Both transformed monocotyledonousand dicotyledonous plants may be obtained in this way. Transgenic planttechnology is for example described in the following publications: SwainWF, 1991, TIBTECH 9: 107-109; Ma JKC et al, 1994, Eur J Immunology 24:131-138; Hiatt A et al, 1992, FEBS Letters 307:71-75; Hein MB et al1991, Biotechnology Progress 7: 455-461; Duering K, 1990, PlantMolecular Biology 15: 281-294.

Examples of genetically modified plants which may be produced includebut are not limited to field crops, cereals, fruit and vegetables suchas: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat,barley, rice, sorghum, mangoes, peaches, apples, pears, strawberries,bananas, melons, tomatoes, potatoes and carrot.

The invention further provides a plant cell containing a gene expressioncassette according to the invention. The gene expression cassette may bestably incorporated in the plant's genome by transformation. Theinvention also provides a plant tissue or a plant comprising such cells,and plants or seeds derived therefrom.

The invention further provides a method for controlling plant geneexpression comprising transforming a plant cell with a plant geneexpression cassette having a bell pepper FIB or CCS promoter operativelylinked to a target gene, whereby the activated promoter drivesexpression of the target gene. The promoter may be activated undercertain spatial, temporal, developmental and/or environmentalconditions.

In order to determine their temporal and spatial expression, thepromoter fragments of the bell pepper FIB and CCS genes are fussed tothe GUS (β-glucuronidase) reporter gene in DNA constructs suitable forplant transformation. GUS accumulation in transgenic plants may then bemonitored.

β-glucuronidase is a bacterial enzyme which catalyses the conversion of5-Methylumbelliferyl glucuronide (MUG) to 4-Methylumbelliferone (MU) andGlucuronic acid. The conversion is measured by way of a fluorometer at365 nm excitation 455 emission. A time course of the reaction can becarried out allowing the conversion rate to be measured in nanoMoles MUformed/mg protein/minute. This activity allows the analysis of geneexpression controlled by the promoter in the transformed plants.

The invention will now be described by way of example and with referenceto the following figures of which;

FIG. 1: Is a diagrammatic map of plasmid pJCCS.Pro.Gus.

FIG. 2: Is a diagrammatic map of plasmid pJFIB.Pro.Gus.

FIG. 3: Graph representing the levels of GUS activity in plantstransformed with construct 1.

FIG. 4: Graph representing the levels of GUS activity in plantstransformed with construct 2.

FIG. 5: Graph representing the fruit development results in plantstransformed with construct 1.

FIG. 6: Graph representing the fruit development results in plantstransformed with construct 2.

FIG. 7: Graph representing the results obtained from floral organs ofplants transformed with constructs 1 and 2.

FIG. 8: Graph representing the levels of GUS activity in developingflowers in plants 15 transformed with construct 1.

FIG. 9: Graph representing the levels of GUS activity in developingflowers in plants transformed with construct 2.

EXAMPLE 1

Isolation of the sequences from the cDNA's.

A pepper genomic fragment (Deruere et al. 1994, Biochem. Biophys Res.Commun. 199 (3) 1144-50)) was characterised by hybridisation to thepepper cDNA for fibrillin (Deruere et al. 1994, The Plant Cell 6 119-33)and by sequence analysis. A 1784 bp fragment upstream of the codingregion was further characterised by sequence analysis. This promotersequence is shown here as SEQ ID No 1. Nucleotides 1782 to 1784 in SEQID No. 1 correspond to the ATG start codon of the GUS gene.

Similarly, a pepper genomic fragment (Deruere et al. 1994, Biochem.Biophys. Res. Commun. 199 (3) 1144-50) was characterised using the CCScDNA (Bouvier et al. 1994, The Plant Journal 6 (1) 45-54 paper) and a2312 bp fragment containing the putative promoter was sequenced and isshown here as SEQ ID No 2. Nucleotides 2310 to 2312 in SEQ ID No 2correspond to the ATG start codon of the GUS gene. Unlike SEQ ID No 1,SEQ ID No 2 is mainly composed of multiple direct repeats.

EXAMPLE 2

SEQ ID No 1 and SEQ ID No 2 were site-directed mutagenised in order tointroduce a coI restriction site at the position of the translationinitiation codon (ccATGg). This col restriction site and a distalrestriction site were used to insert SEQ ID No 1 and SEQ ID No 2,respectively, in front of the GUS gene subcdoned in the plasmidpBluescriptKS and previously modified by site directed mutagenesis inorder to introduce a Ncol restriction site at the position of theinitiation codon. In addition, the nos terminator sequence had beenpreviously inserted downstream of the GUS gene.

These constructs were termed pFib.Pro.Gus (construct 1) and pCCS.Pro.Gus(construct 2), respectively. The whole gene fusion consisting of theputative promoter region fused to the GUS gene and nos terminator wereligated in the plant expression vector JR1Ri to produce pJFib.Pro.Gusand pJCCS.Pro.Gus, respectively.

EXAMPLE 3

Transformation and regeneration of Tomato Explants Transformation wasperformed with the vectors pJFib.Pro.Gus and pJCCS.Pro.Gus. Theseplasmids were transferred to Agrobacterium tumefaciens LBA 4404 (amicroorganism widely available in plant biotechnology) and used totransform tomato stem explants. The transformations produced twoseparate plant lines of primary transformants containing either thepJFib.Pro.Gus construct or the pJCCS.Pro.Gus construct. Thetransformation of tomato stem segments was carried out according tostandard protocols (e.g. Bird et al. (1988), Plant Mol. Biol. 11,651-662). Transformed plants were selected on antibiotic (kanamycin)containing growth media.

EXAMPLE 4

Approximately 50 primary transformants were produced (approximately 25lines containing the pJFib.Pro.Gus or construct 1 and 25 linescontaining the pJCCS.Pro.Gus construct). The presence of the transgeneswas detected by PCR analysis and Southern blot analysis. PCR positiveplants showed 1-3 inserted copies and occasionally up to 7 insertedcopies. Plants were analysed for GUS activity in young leaves and in 4day-post breaker ripening fruits (TABLE 1 and 2) using a fluorimetricassay (Jefferson RA: Plant Mol. Biol. Rep. 5: 387405. 1987). No directcorrelation between transgene copy number and GUS activity was found.Only background GUS activity could be detected in leaves, while high GUSactivity was found in ripening fruits.

Plants containing single insertion were used for fisher analysis. GUSactivity significantly higher than in leaves was also detected invarious flower organs such as petals, anthers and pistil (style andstigma, ovary) and at developmental stages characterised by chromoplastformation (yellow pigmentation). Low GUS activity was detected in fruitpericarp from immature fruits and Gus activity was found to increase atan early mature green stage for FIB and at a late mature green stage forCCS immediately before visible sign of ripening were visible). Gusactivity continued to increase during further ripening. Histochemicalassay for GUS activity showed significant GUS activity in anthers ofboth FIB and CCS plants, as well as in petals for FIB plants (confirmingthat the CCS promoter is more active in anthers than in petals, whilethe FIB promoter is equally active in anthers and petals). Histochemicalassays also showed that both promoters are active in various fruittissues (pericarp, columella, locular tissue).

The following tables show the data obtained from the analysis of theprimary transformants. All the data in the following tables are ameasurement of the GUS activity in nano Moles MU formed per milligramprotein per minute (nM/mg/min).

TABLE 1

Illustrates the GUS activity of tomato leaves and 4 days post “breaker”fruit from plants transformed with construct 1. The table also includesa transformation control (negative control) and a series of positivecontrols including plants transformed with the CaMV-GUS andPolygalacturonase-GUS constructs. The “breaker” stage in tomato fruitripening is an intermediate between the mature green and fully ripestages.

TABLE 1 FRUIT. 4 DAYS POST PLANT NAME LEAVES BREAKER. Transformation0.27 0.80 Control PLANT 1 0.06 0.37 PLANT 2 0.18 0.5 PLANT 3 0.082 1.7PLANT 4 0.38 6.4 PLANT 5 0.15 7.8 PLANT 6 0.094 10 PLANT 7 0.13 40 PLANT8 0.11 97 PLANT 9 0.3 170 PLANT 10 0.087 237 PLANT 11 0.18 240 PLANT 120.125 255 PLANT 13 0.49 320 PLANT 14 0.18 390 PLANT 15 0.31 400 PLANT 160.68 470 PLANT 17 0.34 560 PLANT 18 0.3 600 PLANT 19 0.6 720 PLANT 200.13 720 PLANT 21 0.15 750 PLANT 22 0.14 1000 PLANT 23 0.32 1650 PLANT24 0.26 2200 PG + ve#1 0.1 280 PG + ve#2 1.2 350 PG + ve#3 3.6 460CaMV + ve#1 50 54 CaMV + ve#2 280 170

These data clearly show that the fibrillin promoter is providing highexpression levels of GUS in the fruit of plants 9 through 24 and littleor no expression in the leaves. In particular the expression levels inthe fruit of plants 16 to 24 are higher than that of the positivecontrols which contain the fruit specific PG promoter. As expected theconstitutive CaMV 35S promoter shows high levels of expression of GUS inboth the fruit and leaves providing further evidence that the Fibrillinpromoter is not constitutively expressing throughout the plant.

TABLE 2 Shows the GUS activity in the leaves and 4 days post breakerfruit of plants transformed with construct 2. PLANT FRUIT. 4 DAYS NAMELEAVES POST BREAKER NEGATIVE 0.27 0.80 CONTROL PLANT 25 0.1 0.5 PLANT 260.12 0.94 PLANT 27 0.12 1 PLANT 28 0.075 1.1 PLANT 29 0.17 1.5 PLANT 300.1 3.3 PLANT 31 0.018 12.5 PLANT 32 0.081 34.5 PLANT 33 0.033 76 PLANT34 0.32 150 PLANT 35 0.12 150 PLANT 36 0.38 200 PLANT 37 1.1 240 PLANT38 0.4 270 PLANT 39 0.3 1200 PG + ve#1 0.1 280 PG + ve#2 1.2 350 PG +ve#3 3.6 460 CaMV + ve#1 50 54 CaMV + ve#2 280 170

These data also indicate that the expression levels of GUS are high inthe post breaker fruit and very low in the leaves. Plants 34 to 38 showhigh levels of expression comparable to that of the fruit specificpromoter controls, and plant 39 shows extremely high levels ofexpression.

Results from Tables 1 and 2 clearly show high levels of GUS expressionin the ripening fruit for plants transformed with the FIB and CCSpromoters respectively. These data provide good evidence that thesepromoters are strong and possibly ripening induced.

TABLE 3 Shows the results of the fruit development of two plantstransformed with construct 1. The fruit were analysed for GUS activity avarying stages of development up to and including the mature greenstage. SMALL LATE EARLY LATE PLANT IMMA- IMMA- MATURE MATURE NAME OVARYTURE TURE GREEN GREEN PLANT 24 3.4 1.9  6.2  45  83 PLANT 14 1.9 0.384.4 312 646

The aim of this experiment was to determine at what stage the FIBpromoter was induced. Results indicate that GUS expression is at quitelow levels until the late immature green stage and then begins to risemarkedly at the early mature green stage. Further increase during fruitripening is less pronounced from one fruit to another.

TABLE 4 Shows the results of GUS expression levels during the fruitdevelopment stages of two plants transformed with construct 2. Thisexperiment includes analysis at the post breaker stage. 2 to 4 LATEEARLY LATE DAYS PLANT IMMA- MATURE MATURE POST NAME OVARY TURE GREENGREEN BREAKER PLANT 38 0.54 0.56 0.98 5 60 PLANT 31 0.27 0.5  0.48 2 82

The CCS promoter also appears to increase expression around the maturegreen/breaker fruit ripening stages. It appears that the FIB promoterproduces the strongest expression levels and begins expressing at theearly mature green ripening stage, whereas the CCS promoter expressionlevels are lower and rise around the breaker ripening stage.

TABLE 5 Shows the results obtained from the floral organs from plantstransformed with construct 1 and construct 2. This experiment wasdesigned to test the expression levels in the flowers as they alsocontain areas of chromoplast differentiation which are connected withthe activity of these promoters. The GUS expression levels were assayedin the different parts of the tomato flower, providing the data as setout below. CON- PLANT STRUCT SE- PET- AN- NAME NUMBER LEAVES PALS ALSTHERS PISTIL NEGA- 2 0.27 0.041 0.077 0.036 0.09 TIVE CON- TROL PLANT 20.3 0.16 0.38 18 0.51 39 PLANT 2 0.018 0.15 2 8 No Data 31 PLANT 2 0.40.135 1.7 37.3 0.73 38 PLANT 2 0.12 0.08 0.26 3.6 0.22 35 PLANT 1 0.260.22 33 38 5.4 24 PLANT 1 0.68 0.128 4.6 3.9 1.7 16 PLANT 1 0.18 0.1 2.23.4 0.5 11

These results show that expression levels with the CCS promoter arehigher in the petals, pistils and in particular the anthers whencompared with the leaves and sepals. This is also true for the FIBpromoter, however these plants appear to have higher expression in thepetals and pistils, when compared with the plants containing the CCSpromoter.

TABLE 6 Experiments were conducted on the developing tomato flowers andGUS expression levels were calculated at the different stages offlowering. The first assays being when the immature flower is closed,also when the immature flower is elongating just before opening, whenthe mature flower is completely open and brightly coloured and finallywhen the flower is senescing. IMMA- IMMATURE OPEN AND PLANT TURE BEFOREBRIGHTLY SENES- NAME (CLOSED) (OPENING) COLOURED CING PLANT 11 0.51 0.432.15 5.25 Petals PLANT 11 0.11 0.0925 3.4 12 Anthers PLANT 24 0.6 1.7517.4 19.3 Petals PLANT 24 0.39 3.2 19.7 40.7 Anthers

The data show that the promoter activity increases with the maturity ofthe flowers. This provides further evidence that the FIB promoter isdevelopmentally controlled showing a large rise in promoter activity atthe flower maturity stage.

TABLE 7

Using the same criteria as Table 6, these results show the GUS activityof the petals and the anthers of developing flowers in plantstransformed with construct 2.

TABLE 7 IMMA- IMMATURE OPEN AND PLANT TURE (BEFORE) BRIGHTLY SENES- NAME(CLOSED) (OPENING) COLOURED CING PLANT 31 0.41 0.195 0.195 3.2 PetalsPLANT 31 0.6 0.44 3.2 1.4 Anthers PLANT 38 0.077 0.175 2.75 6.95 PetalsPLANT 38 0.065 0.03 29.1 27.9 Anthers

The data provide further evidence that the CCS promoter isdevelopmentally controlled showing a large rise in promoter activityaround the flower maturity stage.

TABLE 8

This table shows the effects of environmental stress on the expressionlevels in the leaves. This experiment was designed to test the effect ofpromoter activity when environmental stresses are placed upon the plant.The GUS assays were conducted on tomato leaves transformed with eitherconstruct 1 or 2, that had been treated in the following ways; detachedleaf placed in water, detached leaf dehydrated and wounded leaf.

TABLE 8 YOUNG LEAF YOUNG LEAF OLD LEAF OLD LEAF YOUNG DETACHED ANDDETACHED YOUNG DETACHED DETACHED PLANT LEAF PLACED IN AND LEAF OLD LEAFAND PLACED AND OLD LEAF NAME CONTROL WATER DEHYDRATED WOUNDED CONTROL INWATER DEHYDRATED WOUNDED Plant 13 0.08 0.097 0.58 0.1 0.19 0.2 1.1 0.19Plant 14 0.027 0.049 0.13 0.083 Plant 24 0.26 0.75 2.9 0.075 Plant 160.2 0.11 0.38 0.093 Plant 39 0.066 0.068 0.068 0.055 Plant 38 0.086 0.110.092 0.145

These data provide evidence that the FIB promoter can be slightlyinduced by environmental stress such as water defecit but to a muchlower level than that observed during fruit ripening or flowerdevelopment.

2 1 1784 DNA Capsicum annuum 1 gataaatgat cgtattgact tagagtatactggttaaaat tattatatat ctaataattt 60 atttgatgtt ttagtaataa actaccatactgttattttt attttaaact tactgtgaaa 120 gagagaatgc acttcagaaa actaaatatctattacacat catctgaaat ggtcgtgagt 180 gaaccttttt gaatatataa tttaagttgcaaatagagtt atttgaagaa aaaaaaaaag 240 gaaaaagaaa cagtataatg gctccttgtcctctagccca ccaaaaaagg aacaaatatt 300 aatttaatta gaaaaaaaca accttgagcgggtcgaatta gtaggtcttt ccacattcaa 360 attactaatt ttaaaaatta ttgactacttcaagattctt taatcttttt tcctttgtat 420 tgagagagtc atcaaaaagc tcactcgggacgtgaaaaaa aatttgactt gctaattata 480 tctatattta ctaggtaaaa tcctaatcttattatcacat tatcactggg atttttcggc 540 attattctag ctaaatatct tgtgcaattcatgtcctcca ccacaaaaaa aatgcctaat 600 tttacaactt ttttggtatg aaaagagagtaagaaaaaag gaataagaga agtctgattg 660 aaagaaataa ggaaggatga ataaaaaaacaaaagaaaat tctactagaa tttgtatgcc 720 gttggatgtg aatggaaacc aatttttttgtctctcttgt ttgttcaatt ttaaatttcc 780 gccaaacaga cacaaaatga tccttaactccgctttacaa gcgatagtta cgtgtcttcc 840 tctctctccc ttgttgacgt atcttaaaaactccaaacta cccctggatt tttctaatct 900 ttaattaagg attatatata tatatatatatatatatata tatatatata tatatatata 960 tattatgata attaataatt aaatatttgcacatttaaaa gtctatttgg attgacttat 1020 tttagatgtt ttaaagttaa aataacttttaaataatttt agtattcgaa taaactaaga 1080 aaggtgctta taagcacttt atgcctttacactacaatgt aaaaattaag tcaaaagtta 1140 ttaaacgaac ttattagtca aaagttaaagcgaatccaca caagcgctac gcttaatatt 1200 tttttagcca aagcgaatcc aaacaagcgctacgtcaata ttcttttcct tttctttaat 1260 tgaaccatca atcctagatc tcactttctctgacatggga cctaacatta acaatatgag 1320 ttgtggtgtg ataattcgag attccttcaaccctaatagg aagtgctata gtgaaaaatg 1380 agccctccaa tatccgcatt caaatttaatccgactttaa tgtagataca atgtgggaac 1440 cgatgggaaa gaacaaaagt aataagatatacaatatgtt ttcacacata gtgagatcta 1500 gaaagggtag atagtatgcc gttggtatctctatcttaga ggtaaagtag aaaagttgtt 1560 tccaatcgat ccgaactcaa gagaaaaacagtttagccga tgggaaataa agaaaagaaa 1620 aactaattta ggagggtata tatgtctttgcacaaaggct aacctaaagc cctggctcgt 1680 ataaaacgcg atcattacag gattgcaacataaacactca ttcaatcgaa gcttccctgt 1740 taatattatc attttgttcc ttctttattcactcttaaac catg 1784 2 2312 DNA Capsicum annuum 2 gaattcttcc aacagttcgttttttagttt ctgttttggg aagaggagta ctacaaggta 60 ggacctccaa caatcaacaatatctaagtt gcaaaagttt ttgtgcgttt tttagtttct 120 gtttcgagaa gaggaatactacaagttcgt tttttagttt ctgttttggg aagaggagta 180 ctgcaaggta ggacctccaacaattatcaa tatctaaatt gcaaaaattt cagttcgttt 240 ttagtttctg tttcgggaaaaggaatacta caagttcgtt ttttagtttc tattttggga 300 agaggagtac tacaaggtaggacctccaat acctaaattg caaaaatttc agttcgtttt 360 ttagtttcag tttagggaagaggaatacta ctaggtagga cctccaacaa tcatcaatcc 420 agggttgcaa aaatttcagttcgtttttta gtttatgttt gggaagaaga atactacaag 480 gcagtggtgg agctaccttatgattagggg gttcatccga acctccttcg acggaaaatt 540 atactatttt tataagtgaaaattattttt tatgtatata taattgatgt tgaaccccct 600 tcggttagtt catgtatctatattttttta ttttgaaccc cgatgaaaat ttgggctccg 660 ccactgctac aaggtaggacctccaacaat caccaatacc taaattgcaa aaatttcagt 720 ttgcttttta gtttctgttttgggaagagg aatactacaa ggtaggacct ccaacaatca 780 ccaataccta aattgcaacggttttttagt ttctgttttg ggaagaggaa tactacatgg 840 tagggcctcc aacaatcaccaatacctaaa ttgcaaaaat ttcagttcgt attttcgttt 900 ctattttggg aagtggaatagtataaggta ggacctccaa caatcaccaa tacctaaatt 960 gcaaaagttc cgattcattttttagtttct gttttggaaa gagaaatact acaaggtagg 1020 gcctacaaca atcaccagtacctaaattgt aaaaatttca gttcgttttt tagtttctat 1080 tttgagaaga ggaatgctacaaggtagggc ctacaacaat caccagtacc taaattgtaa 1140 aaatttcagt tcgttttttagtttctgttt tgggaagagg aatactacaa ggtagggcct 1200 ccaacaatca gcaatacctaaattacaaaa atttcaattc gttttttagt ttctgttttg 1260 ggaagaggaa tactacaaggcagtggcgga gctaccttat gattaggggt tcatccgaac 1320 ctccttcgac ggaaaattatactatttttg tatagtaaaa attatttttt atgtatatat 1380 aattgatgtt gaacccccttcggttagttt gtgtatctat atttttttat tttgaacctc 1440 cttgataaaa aattttgactccgccattgc tacaaggtag aacctccaac aatcaccaat 1500 acctaaattg caaaaatttcagttcgtttt ttaatttctg ttttgggaag aggaatacta 1560 caggcctcca acaatcaccaatacctaaat tgcaaaattt tagtttgttt tttagtttct 1620 gttttgggaa gaggaatactacaaggtaag gcctccaaca atcaccaata cctaaattgc 1680 aaaaatttca gttcgtattttcgtttctat tttgggaagt ggaatagtat aaggtaggac 1740 ctccaacaat caccaatacctaaattgcaa aagttccgat tcatttttta gtttctgttt 1800 tggaaagaga aatactacaaggtagggcct ccaacaatca ccagtaccta aattgtaaaa 1860 atttcagttc gttttttagtttctattttg ggaagtggaa tagtataagg taggacctcc 1920 aacaatcacc aatacctaaattgcaaaagt tccgattctt tttttagttt ctgttttgga 1980 aagagaaata ctacaagataggaccttcaa caatcaccaa tacctaaatt gcaaaaactt 2040 cagttcattt tttagtttctgttttgggaa gaagaatact tcaaggtaac aatcaccaat 2100 acctaaatta aaaatttcagttagtttttt agtttctgtt tttgggaaga ggaatacttt 2160 cttttgctat ataaagccaaagtaggtacc tataagcatc aatattttgt attgcttagt 2220 gattccccta gttcggtatttcattttttt tcactatact atatcacctc ctctcataaa 2280 tagccattat aaatcttgcattttctctaa tg 2312

What is claimed is:
 1. A DNA sequence encoding a Capsicum anuum fibrillin gene promoter capable of driving gene expression in plants, wherein the DNA sequence comprising the sequence shown in SEQ ID NO:
 1. 2. A DNA construct comprising a promoter as claimed in claim 1 operatively linked to a transcribable DNA region and a transcription termination signal.
 3. A transgenic plant, seed or any other form of regenerant having stably incorporated within its genome a DNA construct as claimed in claim
 2. 4. A plant according to claim 3 in which the said plant is a tomato plant.
 5. A method for controlling gene expression in a plant comprising transforming a plant cell with a plan gene expression cassette having a promoter as claimed in claim 1 operatively linked to a target gene. 